Transition metal complexes with (pyridyl)imidazole ligands

ABSTRACT

Novel transition metal complexes of iron, cobalt, ruthenium, osmium, and vanadium are described. The transition metal complexes can be used as redox mediators in enzyme-based electrochemical sensors. The transition metal complexes include substituted or unsubstituted (pyridyl)imidazole ligands. Transition metal complexes attached to polymeric backbones are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional PatentApplication Serial No. 60/290,537 of Fei Mao, filed on May 11, 2001 andentitled “Transition Metal Complexes with (Pyridyl)imidazole Ligands”,which is incorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

[0002] This invention relates to transition metal complexes with(pyridyl)imidazole ligands. In addition, the invention relates to thepreparation of the transition metal complexes and to the use of thetransition metal complexes as redox mediators.

BACKGROUND OF THE INVENTION

[0003] Enzyme-based electrochemical sensors are widely used in thedetection of analytes in clinical, environmental, agricultural andbiotechnological applications. Analytes that can be measured in clinicalassays of fluids of the human body include, for example, glucose,lactate, cholesterol, bilirubin and amino acids. Levels of theseanalytes in biological fluids, such as blood, are important for thediagnosis and the monitoring of diseases.

[0004] Electrochemical assays are typically performed in cells with twoor three electrodes, including at least one measuring or workingelectrode and one reference electrode. In three electrode systems, thethird electrode is a counter-electrode. In two electrode systems, thereference electrode also serves as the counter-electrode. The electrodesare connected through a circuit, such as a potentiostat. The measuringor working electrode is a non-corroding carbon or metal conductor. Uponpassage of a current through the working electrode, a redox enzyme iselectrooxidized or electroreduced. The enzyme is specific to the analyteto be detected, or to a product of the analyte. The turnover rate of theenzyme is typically related (preferably, but not necessarily, linearly)to the concentration of the analyte itself, or to its product, in thetest solution.

[0005] The electrooxidation or electroreduction of the enzyme is oftenfacilitated by the presence of a redox mediator in the solution or onthe electrode. The redox mediator assists in the electricalcommunication between the working electrode and the enzyme. The redoxmediator can be dissolved in the fluid to be analyzed, which is inelectrolytic contact with the electrodes, or can be applied within acoating on the working electrode in electrolytic contact with theanalyzed solution. The coating is preferably not soluble in water,though it may swell in water. Useful devices can be made, for example,by coating an electrode with a film that includes a redox mediator andan enzyme where the enzyme is catalytically specific to the desiredanalyte, or its product. In contrast to a coated redox mediator, adiffusional redox mediator, which can be soluble or insoluble in water,functions by shuttling electrons between, for example, the enzyme andthe electrode. In any case, when the substrate of the enzyme iselectrooxidized, the redox mediator transports electrons from thesubstrate-reduced enzyme to the electrode; and when the substrate iselectroreduced, the redox mediator transports electrons from theelectrode to the substrate-oxidized enzyme.

[0006] Recent enzyme-based electrochemical sensors have employed anumber of different redox mediators such as monomeric ferrocenes,quinoid compounds including quinines (e.g., benzoquinones), nickelcyclamates, and ruthenium amines. For the most part, these redoxmediators have one or more of the following limitations: the solubilityof the redox mediators in the test solutions is low, their chemical,light, thermal, and/or pH stability is poor, or they do not exchangeelectrons rapidly enough with the enzyme or the electrode or both. Somemediators with advantageous properties are difficult to synthesize.Additionally, the redox potentials of some of these reported redoxmediators are so oxidizing that at the potential at which the reducedmediator is electrooxidized on the electrode, solution components otherthan the analyte are also electrooxidized. Some other of these reportedredox mediators are so reducing that solution components, such as, forexample, dissolved oxygen, are also rapidly electroreduced. As a result,the sensor utilizing the mediator is not sufficiently specific.

SUMMARY OF THE INVENTION

[0007] The present invention is directed to novel transition metalcomplexes. The present invention is also directed to the use of thecomplexes as redox mediators. The preferred redox mediators typicallyexchange electrons rapidly with enzymes and electrodes, are stable, canbe readily synthesized, and have a redox potential that is tailored forthe electrooxidation of analytes, such as glucose for example.

[0008] One embodiment of the invention is a transition metal complexhaving the general formula set forth below.

[0009] In this general formula, M is cobalt, iron, ruthenium, osmium, orvanadium; c is an integer selected from −1 to −5, 0, or +1 to +5indicating a positive, neutral, or negative charge; X represents atleast one counter ion; d is an integer from 0 to 5 representing thenumber of counter ions, X; L and L′ are independently selected from thegroup consisting of:

[0010] and L₁ and L₂ are other ligands. In the formula for L and L′, R′₁is a substituted or an unsubstituted alkyl, alkenyl, or aryl group.Generally, R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d) are independently—H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂,or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino,alkylthio, aikenyl, aryl, or alkyl.

[0011] The transition metal complexes of the present invention areeffectively employed as redox mediators in electrochemical sensors,given their very fast kinetics. More particularly, when a transitionmetal complex of this invention is so employed, rapid electron exchangebetween the transition metal complex and the enzyme and/or the workingelectrode in the sensor device occurs. This electron exchange issufficiently rapid to facilitate the transfer of electrons to theworking electrode that might otherwise be transferred to anotherelectron scavenger in the system. The fast kinetics of the mediator isgenerally enhanced when L₂ of a mediator of the formula provided aboveis a negatively charged ligand.

[0012] The transition metal complexes of the present invention are alsoquite stable. For example, when such a complex is used as a mediator inan electrochemical sensor, the chemical stability is generally such thatthe predominant reactions in which the mediator participates are theelectron-transfer reaction between the mediator and the enzyme and theelectrochemical redox reaction at the working electrode. The chemicalstability may be enhanced when a mediator of the formula provided above,wherein L₂ is a negatively charged ligand, has a “bulky” chemicalligand, L₁, that shields the redox center, M, and thereby reducesundesirable chemical reactivity beyond the desired electrochemicalactivity.

[0013] The electrochemical stability of the transition metal complexesof the present invention is also quite desirable. For example, when sucha complex is used as a mediator in an electrochemical sensor, themediator is able to operate in a range of redox potentials at whichelectrochemical activity of common interfering species is minimized andgood kinetic activity of the mediator is maintained.

[0014] Thus, the present invention provides novel transition metalcomplexes that are particularly useful as redox mediators inelectrochemical sensing applications. The advantageous properties andcharacteristics of the transition metal complexes of the presentinvention make them ideal candidates for use in the electrochemicalsensing of glucose, an application of particular importance in thetreatment of diabetes in human populations.

DETAILED DESCRIPTION

[0015] When used herein, the definitions set forth below in quotationsdefine the stated term.

[0016] The term “alkyl” includes linear or branched, saturated aliphatichydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, tert-butyl and the like. Unless otherwise noted, theterm “alkyl” includes both alkyl and cycloalkyl groups.

[0017] The term “alkoxy” describes an alkyl group joined to theremainder of the structure by an oxygen atom. Examples of alkoxy groupsinclude methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, andthe like. In addition, unless otherwise noted, the term ‘alkoxy’includes both alkoxy and cycloalkoxy groups.

[0018] The term “alkenyl” describes an unsaturated, linear or branchedaliphatic hydrocarbon having at least one carbon-carbon double bond.Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl,1-butenyl, 2-methyl-1-propenyl, and the like.

[0019] A “reactive group” is a functional group of a molecule that iscapable of reacting with another compound to couple at least a portionof that other compound to the molecule. Reactive groups include carboxy,activated ester, sulfonyl halide, sulfonate ester, isocyanate,isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine,acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine,alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate,halotriazine, imido ester, maleimide, hydrazide, hydroxy, andphoto-reactive azido aryl groups. Activated esters, as understood in theart, generally include esters of succinimidyl, benzotriazolyl, or arylsubstituted by electron-withdrawing groups such as sulfo, nitro, cyano,or halo groups; or carboxylic acids activated by carbodiimides.

[0020] A “substituted” functional group (e.g., substituted alkyl,alkenyl, or alkoxy group) includes at least one substituent selectedfrom the following: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, —NH₂, alkylamino,dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido,hydrazino, alkylthio, alkenyl, and reactive groups.

[0021] A “biological fluid” is any body fluid or body fluid derivativein which the analyte can be measured, for example, blood, interstitialfluid, plasma, dermal fluid, sweat, and tears.

[0022] An “electrochemical sensor” is a device configured to detect thepresence of or measure the concentration or amount of an analyte in asample via electrochemical oxidation or reduction reactions. Thesereactions typically can be transduced to an electrical signal that canbe correlated to an amount or concentration of analyte.

[0023] A “redox mediator” is an electron transfer agent for carryingelectrons between an analyte or an analyte-reduced or analyte-oxidizedenzyme and an electrode, either directly, or via one or more additionalelectron transfer agents. Redox mediators that include a polymericbackbone may also be referred to as “redox polymers”.

[0024] “Electrolysis” is the electrooxidation or electroreduction of acompound either directly at an electrode or via one or more electrontransfer agents (e.g., redox mediators or enzymes).

[0025] The term “reference electrode” includes both a) referenceelectrodes and b) reference electrodes that also function as counterelectrodes (i.e., counter/reference electrodes), unless otherwiseindicated.

[0026] The term “counter electrode” includes both a) counter electrodesand b) counter electrodes that also function as reference electrodes(i.e., counter/reference electrodes), unless otherwise indicated.

[0027] Generally, the present invention relates to transition metalcomplexes of iron, cobalt, ruthenium, osmium, and vanadium having(pyridyl)imidazole ligands. The invention also relates to thepreparation of the transition metal complexes and to the use of thetransition metal complexes as redox mediators. In at least someinstances, the transition metal complexes have one or more of thefollowing characteristics: redox potentials in a particular range, theability to exchange electrons rapidly with electrodes, the ability torapidly transfer electrons to or rapidly accept electrons from an enzymeto accelerate the kinetics of electrooxidation or electroreduction of ananalyte in the presence of an enzyme or another analyte-specific redoxcatalyst. For example, a redox mediator may accelerate theelectrooxidation of glucose in the presence of glucose oxidase orPQQ-glucose dehydrogenase, a process that can be useful for theselective assay of glucose in the presence of other electrochemicallyoxidizable species. Some embodiments of the invention may be easier ormore cost-effective to make synthetically or use more widely availableor more cost-effective reagents in synthesis than other transition metalredox mediators.

[0028] Compounds having Formula 1, set forth below, are examples oftransition metal complexes of the present invention.

[0029] M is a transition metal and is typically iron, cobalt, ruthenium,osmium, or vanadium. Ruthenium and osmium are particularly suitable forredox mediators.

[0030] L and L′ are each bidentate, substituted or unsubstituted2-(2-pyridyl)imidazole ligands having the Structure 2 set forth below.

[0031] In Structure 2, R′₁ is a substituted or an unsubstituted aryl,alkenyl, or alkyl. Generally, R′₁ is a substituted or an unsubstitutedC1-C12 alkyl or alkenyl, or an aryl, such as phenyl, optionallysubstituted with a substituent selected from a group consisting of —Cl,—F, —CN, amino, carboxy, C1-C6 alkyl, C1-C6 alkylthio, C1-C6 alkylamino,C1-C6 dialkylamino, C1-C6 alkylaminocarbonyl, C1-C6 alkoxy, C1-C6alkoxycarbonyl, and C1-C6 alkylcarboxamido. R′₁ is typically methyl or aC1-C12 alkyl that is optionally substituted with a reactive group, or anaryl optionally substituted with C1-C2 alkyl, C1-C2 alkoxy, —Cl, or —F.

[0032] Generally, R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d) areindependently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂,—SH, —OH, —NH₂, substituted or unsubstituted alkoxylcarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R_(c) and R_(d) in combination and/or R′₃ and R′₄ incombination can form a saturated or unsaturated 5- or 6-membered ring.Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl oraryl portions of any of the substituents are optionally substituted by—F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium (except onaryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally,R′₃, R′₄, R_(a), R_(b), R_(c) and R_(d) are independently —H orunsubstituted alkyl groups. Typically, R_(a) and R_(c) are —H and R′₃,R′₄, R_(b), and R_(d) are —H or methyl.

[0033] Preferably, the L and L′ ligands are the same. Herein, referencesto L and L′ may be used interchangeably.

[0034] In Formula 1, c is an integer indicating the charge of thecomplex. Generally, c is an integer selected from −1 to −5 or +1 to +5indicating a positive or negative charge or 0 indicating a neutralcharge. For a number of osmium complexes, c is +1, +2, or +3.

[0035] X represents counter ion(s). Examples of suitable counter ionsinclude anions, such as halide (e.g., fluoride, chloride, bromide oriodide), sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate,and cations (preferably, monovalent cations), such as lithium, sodium,potassium, tetralkylammonium, and ammonium. Preferably, X is a halide,such as chloride. The counter ions represented by X are not necessarilyall the same.

[0036] d represents the number of counter ions and is typically from 0to 5.

[0037] L₁ and L₂ are ligands attached to the transition metal via acoordinative bond. L₁ and L₂ are monodentate ligands, at least one ofwhich is a negatively charged monodentate ligand. While L₁ and L₂ may beused interchangeably, L₂ is generally referred to as a negativelycharged ligand merely by way of convenience. Herein, the term“negatively charged ligand” is defined as a ligand in which thecoordinating atom itself is negatively charged so that on coordinationto a positively charged metal, the negative charge is neutralized. Forexample, a halide such as chloride or fluoride meets the presentdefinition while a pyridine ligand bearing a negatively chargedsulfonate group does not because the sulfonate group does notparticipate in coordination. Examples of negatively charged ligandsinclude, but are not limited to, —F, —Cl, —Br, —I, —CN, —SCN, —OH,alkoxy, alkylthio, and phenoxide. Typically, the negatively chargedmonodentate ligand is a halide.

[0038] Examples of other suitable monodentate ligands include, but arenot limited to, H₂O, NH₃, alkylamine, dialkylamine, trialkylamine, orheterocyclic compounds. The alkyl or aryl portions of any of the ligandsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. Any alkyl portions of themonodentate ligands generally contain 1 to 12 carbons. More typically,the alkyl portions contain 1 to 6 carbons. In other embodiments, themonodentate ligands are heterocyclic compounds containing at least onenitrogen, oxygen, or sulfur atom. Examples of suitable heterocyclicmonodentate ligands include imidazole, pyrazole, oxazole, thiazole.triazole, pyridine, pyrazine and derivatives thereof. Suitableheterocyclic monodentate ligands include substituted and unsubstitutedimidazole and substituted and unsubstituted pyridine having the generalFormulas 3 and 4, respectively, as set forth below.

[0039] With regard to Formula 3, R₇ is generally a substituted orunsubstituted alkyl, alkenyl, or aryl group. Generally, R₇ is asubstituted or unsubstituted C1 to C12 alkyl or alkenyl, or an aryl,such as phenyl, optionally substituted with a substituent selected froma group consisting of —Cl, —F, —CN, amino, carboxy, C1-C6 alkyl, C1-C6alkylthio, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6alkylaminocarbonyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, and C1-C6alkylcarboxamido. R₇ is typically methyl or a C1-C12 alkyl that isoptionally substituted with a reactive group, or an aryl optionallysubstituted with C1-C2 alkyl, C1-C2 alkoxy, —Cl, or —F.

[0040] Generally, R₈, R₉ and R₁₀ are independently —H, —F, —Cl, —Br, —I,—NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl.Alternatively, R₉ and R₁₀, in combination, form a fused 5- or 6-memberedring that is saturated or unsaturated. The alkyl portions of thesubstituents generally contain 1 to 12 carbons and typically contain 1to 6 carbon atoms. The alkyl or aryl portions of any of the substituentsare optionally substituted by —F, —Cl, —Br, —I, alkylamino,dialkylamino, trialkylammonium (except on aryl portions), alkoxy,alkylthio, aryl, or a reactive group. In some embodiments, R₈, R₉ andR₁₀ are —H or substituted or unsubstituted alkyl. Preferably, R₈, R₉ andR₁₀ are —H.

[0041] With regard to Formula 4, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ areindependently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —OH, —NH₂,alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, oralkyl. The alkyl or aryl portions of any of the substituents areoptionally substituted by —F, —Cl, —Br, —I, alkylamino, dialkylamino,trialkylammonium (except for aryl portions), alkoxy, alkylthio, aryl, ora reactive group. Generally, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are —H, methyl,C1-C2 alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 loweralkyl substituted with a reactive group.

[0042] One example includes R₁₁ and R₁₅ as —H, R₁₂ and R₁₄ as the sameand —H or methyl, and R₁₃ as —H, C1 to C12 alkoxy, —NH₂, C1 to C12alkylamino, C2 to C24 dialkylamino, hydrazino, C1 to C12 alkylhydrazino,hydroxylamino, C1 to C12 alkoxyamino, C1 to C12 alkylthio, or C1 to C12alkyl. The alkyl or aryl portions of any of the substituents areoptionally substituted by —F, —Cl, —Br, −I, alkylamino, dialkylamino,trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, ora reactive group.

[0043] Examples of suitable transition metal complexes include[Os[1-methyl-2-(2-pyridyl)imidazole]₂(1-methylimidazole)Cl]²⁺2Cl— (alsowritten as [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—) where L₁ is

[0044] L₂ is Cl; c is +2; d is 2; X is Cl—; and L and L′ are

[0045] The transition metal complexes of Formula I also includetransition metal complexes that are coupled to a polymeric backbonethrough one or more of L, L′, L₁, and L₂. In some embodiments, thepolymeric backbone has at least one functional group that acts as aligand of the transition metal complex. Such polymeric backbonesinclude, for example, poly(4-vinylpyridine) and poly(N-vinylimidazole)in which the pyridine and imidazole groups, respectively, can act asmonodentate ligands of the transition metal complex. In otherembodiments, the transition metal complex can be the reaction productbetween a reactive group on a precursor polymer and a reactive group ona ligand of a precursor transition metal complex (such as complex ofFormula 1 where one of L, L′, L₁, and L₂ includes a reactive group, asdescribed above). Suitable precursor polymers include, for example,poly(acrylic acid) (Formula 7), styrene/maleic anhydride copolymer(Formula 8), methylvinylether/maleic anhydride copolymer (GANTREZpolymer) (Formula 9), poly(vinylbenzylchloride) (Formula 10),poly(allylamine) (Formula 11), polylysine (Formula 12),carboxy-poly(vinylpyridine) (Formula 13), and poly(sodium 4-styrenesulfonate) (Formula 14). The numbers n, n′ and n″ appearing variously inthese formulas may vary widely. Merely by way of example, in Formula 13,[n′/(n′+n″)]×100% is preferably from about 5% to about 15%.

[0046] Alternatively, the transition metal complex can have one or morereactive group(s) for immobilization or conjugation of the complexes toother substrates or carriers, examples of which include, but are notlimited to, macromolecules (e.g., enzymes) and surfaces (e.g., electrodesurfaces).

[0047] For reactive attachment to polymers, substrates, or othercarriers, the transition metal complex precursor includes at least onereactive group that reacts with a reactive group on the polymer,substrate, or carrier. Typically, covalent bonds are formed between thetwo reactive groups to generate a linkage. Examples of such reactivegroups and resulting linkages are provided in Table 1, below. Generally,one of the reactive groups is an electrophile and the other reactivegroup is a nucleophile. TABLE 1 Examples of Reactive Groups andResulting Linkages First Reactive Group Second Reactive Group ResultingLinkage Activated ester* Amine Carboxamide Acrylamide Thiol ThioetherAcyl azide Amine Carboxamide Acyl halide Amine Carboxamide Carboxylicacid Amine Carboxamide Aldehyde or ketone Hydrazine Hydrazone Aldehydeor ketone Hydroxyamine Oxime Alkyl halide Amine Alkylamine Alkyl halideCarboxylic acid Carboxylic ester Alkyl halide Imidazole ImidazoliumAlkyl halide Pyridine Pyridinium Alkyl halide Alcohol/phenol Ether Alkylhalide Thiol Thioether Alkyl sulfonate Thiol Thioether Alkyl sulfonatePyridine Pyridinium Alkyl sulfonate Imidazole Imidazolium Alkylsulfonate Alcohol/phenol Ether Anhydride Alcohol/phenol Ester AnhydrideAmine Carboxamide Aziridine Thiol Thioether Aziridine Amine AlkylamineAziridine Pyridine Pyridinium Epoxide Thiol Thioether Epoxide AmineAlkylamine Epoxide Pyridine Pyridinium Halotriazine Amine AminotriazineHalotriazine Alcohol Triazinyl ether Imido ester Amine AmidineIsocyanate Amine Urea Isocyanate Alcohol Urethane Isothiocyanate AmineThiourea Maleimide Thiol Thioether Sulfonyl halide Amine Sulfonamide

[0048] Transition metal complexes of the present invention can besoluble in water or other aqueous solutions, or in organic solvents. Ingeneral, the transition metal complexes can be made soluble in eitheraqueous or organic solvents by having an appropriate counter ion orions, X. For example, transition metal complexes with small counteranions, such as F—, Cl—, and Br—, tend to be water soluble. On the otherhand, transition metal complexes with bulky counter anions, such as I—,BF₄— and PF₆—, tend to be soluble in organic solvents. Preferably, thesolubility of transition metal complexes of the present invention isgreater than about 0.1 M (moles/liter) at 25° C. for a desired solvent.

[0049] The transition metal complexes discussed above are useful asredox mediators in electrochemical sensors for the detection of analytesin biofluids. The use of transition metal complexes as redox mediatorsis described, for example, in U.S. Pat. Nos. 5,262,035, 5,320,725,5,365,786, 5,593,852, 5,665,222, 5,972,199, 6,134,161, 6,143,164,6,175,752 and 6,338,790 and U.S. patent application Ser. No. 09/434,026,all of which are incorporated herein by reference. The transition metalcomplexes described herein can typically be used in place of thosediscussed in the references listed above, although the results of suchuse will be significantly enhanced given the particular properties ofthe transition metal complexes of the present invention, as furtherdescribed herein.

[0050] In general, the redox mediator of the present invention isdisposed on or in proximity to (e.g., in a solution surrounding) aworking electrode. The redox mediator transfers electrons between theworking electrode and an analyte.. In some preferred embodiments, anenzyme is also included to facilitate the transfer. For example, theredox mediator transfers electrons between the working electrode andglucose (typically via an enzyme) in an enzyme-catalyzed reaction ofglucose. Redox polymers are particularly useful for formingnon-leachable coatings on the working electrode. These can be formed,for example, by crosslinking the redox polymer on the working electrode,or by crosslinking the redox polymer and the enzyme on the workingelectrode.

[0051] Transition metal complexes can enable accurate, reproducible andquick or continuous assays. Transition metal complex redox mediatorsaccept electrons from, or transfer electrons to, enzymes or analytes ata high rate and also exchange electrons rapidly with an electrode.Typically, the rate of self exchange, the process in which a reducedredox mediator transfers an electron to an oxidized redox mediator, israpid. At a defined redox mediator concentration, this provides for morerapid transport of electrons between the enzyme (or analyte) andelectrode, and thereby shortens the response time of the sensor.Additionally, the novel transition metal complex redox mediators aretypically stable under ambient light and at the temperatures encounteredin use, storage and transportation. Preferably, the transition metalcomplex redox mediators do not undergo chemical change, other thanoxidation and reduction, in the period of use or under the conditions ofstorage, though the redox mediators can be designed to be activated byreacting, for example, with water or the analyte.

[0052] The transition metal complex can be used as a redox mediator incombination with a redox enzyme to electrooxidize or electroreduce theanalyte or a compound derived of the analyte, for example by hydrolysisof the analyte. The redox potentials of the redox mediators aregenerally more positive (i.e. more oxidizing) than the redox potentialsof the redox enzymes when the analyte is electrooxidized and morenegative when the analyte is electroreduced. For example, the redoxpotentials of the preferred transition metal complex redox mediatorsused for electrooxidizing glucose with glucose oxidase or PQQ-glucosedehydrogenase as enzyme is between about −200 mV and +200 mV versus aAg/AgCl reference electrode, and the most preferred mediators have redoxpotentials between about −200 mV and about +100 mV versus a Ag/AgClreference electrode.

Examples of Syntheses of Transition Metal Complexes

[0053] Examples showing the syntheses of various transition metalcomplexes that are useful as redox mediators are provided below. Unlessindicated otherwise, all of the chemical reagents are available fromAldrich Chemical Co. (Milwaukee, Wis.) or other sources. Numericalfigures provided are approximate.

EXAMPLE 1 Synthesis of [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—

[0054] By way of illustration, an example of the synthesis of[Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—, as illustrated below, is now provided.

Synthesis of 2-(2-pyridyl)imidazole

[0055] A solution of pyridine-2-carboxaldehyde (151.4 g, 1.41 moles) andglyoxal (40% in H₂O, 205 mL, 1.79 moles) in 300 mL of ethanol (EtOH) ina three-necked 1 L round-bottom flask fitted with a thermometer and anaddition funnel was stirred in an ice bath. When the solution was cooledto below 5° C., concentrated NH₄OH (28-30%, 482 mL, 3.93 moles) wasadded dropwise through the addition funnel. The rate of the addition wascontrolled so that the temperature of the solution was maintained atbelow 5° C. After the addition, the stirring of the solution wascontinued in the ice bath for one hour and then at room temperatureovernight. During the stirring process, the solution changed from lightyellow to dark brown.

[0056] The solution was transferred to a 2 L round bottom flask and theEtOH solvent was removed by rotary evaporation. The resulting darkviscous material was transferred to a 4 L beaker with 700 mL of EtOAc.500 mL of saturated NaCl was added and the mixture was stirred for 2hours. The solution was poured into a 2 L separation funnel and a darktarry material was discarded. The organic layer was separated from thesolution and the aqueous layer was extracted several times with EtOAc(500 mL EtOAc per extraction). The organic layer was then dried withanhydrous Na₂SO₄ overnight, whereupon the resulting mixture was gravityfiltered, the Na₂SO₄ was washed with EtOAc (4×50 mL), and the solutionwas concentrated to about 300-400 mL by rotary evaporation. Theconcentrated solution was transferred to a 1 L Erlenmeyer flask and thevolume was adjusted with more EtOAc to about 400-500 mL, as necessary.The solution stood at 4° C. for 1-2 days to form large amber crystals.The crystals were collected by suction filtration and washed with coldEtOAc (20-30 mL). The filtrate contained a large amount of product, sofurther concentration and crystallization procedures were performed. Thecrystals were combined and dried at 40-45° C. under high vacuum for 2days. The yield of 2-(2-pyridyl)imidazole was about 75 g.

Synthesis of 1-methyl-2-(2-pyridyl)imidazole

[0057] Pyridine-2-carboxaldehyde (50.5 g, 0.47 moles) and glyoxal (40%in H₂O, 68.3 mL, 0.60 moles) in 100-150 mL of ethanol (EtOH) in athree-necked 1 L round-bottom flask fitted with a thermometer and anaddition funnel were stirred in an ice bath. When the solution wascooled to below 5° C., concentrated NH₄OH (28-30%, 161 mL, 1.31 moles)was added dropwise through the addition funnel. The rate of the additionwas controlled so that the temperature of the solution was maintained atbelow 5° C. After the addition, the stirring of the solution wascontinued in the ice bath for one hour and then at room temperatureovernight. During the stirring process, the solution changed from lightyellow to dark brown.

[0058] The solution was transferred to a 1 L round bottom flask and theEtOH and H₂O solvent was removed by rotary evaporation at 50° C. Theresulting material was dried further at about 50° C. under high vacuumfor 24 hours and then dissolved in anhydrous dimethyl formamide (DMF),whereupon the solution was transferred with further DMF (total DMF450-500 mL) to a three-necked 1 L round bottom flask equipped with areflux condenser, and then stirred. Sodium t-butoxide (48.9 g, 0.51moles) was added quickly via a funnel to obtain, with continued stirringfor about 1 hour, a dark brown homogeneous solution. Methyl iodide (34.5mL, 0.56 moles) was then added dropwise via an addition funnel over1.5-2 hours, resulting in a white precipitate of NaI. The mixture wasstirred at room temperature overnight, its color changing from darkbrown to light brown. The mixture was then poured into a beakercontaining 1.5 mL of EtOAc and suction-filtered using a Buchner funnelto remove the NaI precipitate. The precipitate was washed withadditional EtOAc (3×100 mL). The filtrate was transferred. to a 2 Lround bottom flash and rotary evaporated to remove the EtOAc.

[0059] The resulting viscous material was transferred to a 1 L beakerwith a minimum amount of EtOAc, which was then removed by rotaryevaporation. The remaining DMF was removed by vacuum distillation usinga low vacuum diaphragm pump and an oil bath. Upon complete removal ofthe DMF, the product was distilled at 100-110° C. under high vacuum. Theyield of 1-methyl-2-(2-pyridyl)imidazole was about 36 g.

Synthesis of Os(Py-MIM)₂Cl₂

[0060] 1-methyl-2-(2-pyridyl)imidazole (3.4 g, 21.4 mmoles) and ammoniumhexachloroosmiate (IV) (4.7 g, 10.7 mmoles) were combined with anhydrousethylene glycol (86 mL) in a three-necked 250 mL round bottom flask,fitted with a reflux condenser, immersed in a temperature-controlled oilbath. The reaction mixture was degassed with N₂ for about 15 minutes.The mixture was stirred under N₂ while the heater was turned on to heatthe oil bath, and the reaction proceeded at 130° C. for 2 hours andsubsequently at 140° C. for about 28 hours until an intermediate thatwas formed in the reaction was completely converted to the finalproduct. The solution was cooled to room temperature and thensuction-filtered through a fitted funnel into a three-necked 250 mLround bottom flask, whereupon a small amount of orange precipitate leftin the funnel was discarded. The solution (solution A) was then degassedwith N₂ for 15 minutes and kept under N₂.

[0061] Deionized H₂O (320 mL) was then degassed with N₂ in athree-necked 500 mL round bottom flask cooled in an ice/water bath andequipped with a thermometer. After 15 minutes of degassing, sodiumhydrosulfite (85%, 9.31 g, 53.5 mmoles) under N₂ was added immediatelyand degassing continued for another 10-15 minutes. The temperature ofthe solution (solution B) was below 5° C. Solution A was then added viaa canula to solution B under rapid stirring for about 0.5 hour to form afine dark purple precipitate of Os(Py-MIM)₂Cl₂. Stirring continued underN₂ for another 0.5 hour. The resulting suspension was suction-filteredthrough a 0.4 or 0.3 micron Nylon membrane. The suspension wastransferred to the suction funnel via a canula under nitrogen tominimize air exposure. The dark purple precipitate was then washed witha minimum of ice cold water (2×5 mL). The precipitate was immediatelydried by lyophilization for at least 24 hours. The yield ofOs(Py-MIM)₂Cl₂ was about 5.6 g.

Synthesis of [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl—

[0062] Anhydrous ethanol (1 L) in a 2-L three-necked round bottom flaskfitted with a reflux condenser was degassed with N₂ for 15 minutes.Os(Py-MIM)₂Cl₂ (3.1 g, 5.35 mmoles) was added quickly under N₂ via afunnel. The suspension was stirred and heated to reflux.1-methylimidazole (0.43 mL, 5.35 mmoles) was then added at once via asyringe. Reflux continued until the reaction was completed. During thereaction, the solution changed from dark brown to purple-brown. Thesolution was cooled to room temperature and then suction-filteredthrough a fitted funnel. The solvent was then removed by rotaryevaporation to give the crude product in its reduced form.

[0063] The product was transferred with 30-50 mL H₂O to a 400 mL beakercontaining about 40 mL AG1×4 chloride resin from Bio-Rad, or preferably,80 mL Dowex-1-chloride from Aldrich. The mixture was stirred in open airfor about 24 hours to convert Os(II) to Os(III). The mixture was thensuction-filtered and the resin was washed with H₂O (5×30 mL). Thecombined filtrate was concentrated to about 50 mL by rotary evaporationat 35° C. under vacuum.

[0064] The solution was loaded onto a LH-20 column (2″×22″), which waseluted with H₂O. 50 mL fractions were collected and analyzed by CV tofind the major purple-brown band associated with the product. Fractionscontaining pure product were collected and concentrated by rotaryevaporation to about 150 mL. The solution was then freeze-dried to givethe product. The yield of [Os(Py-MIM)₂(MM)Cl]²⁺2Cl— was about 2.4 g.

[0065] As described herein, [Os(Py-MIM)₂(MIM)Cl]²⁺2Cl— is a transitionmetal complex that is particularly useful as a redox mediator.

EXAMPLE 2 Synthesis of 1-phenyl-2-(2-pyridyl)imidazole

[0066] Further by way of illustration, an example of the synthesis of1-phenyl-2-(2-pyridyl)imidazole, as illustrated below, is now provided.The example demonstrates how a 1-aryl-substituted 2-(2-pyridyl)imidazoleis made from 1-(2-pyridyl)imidazole or its derivative, and aniodobenzene derivative (as illustrated) or a bromobenzene derivative.

[0067] 2-(2-pyridyl)imidazole (6.91 g), iodobenzene (11.47 g), Cs₂CO₃(25 g), and copper powder (15 g) were mixed in 60 mL anhydrous DMF in a250 mL round bottom flask equipped with a magnetic stirrer and a refluxcondesner. The mixture is degassed with N₂ for 15 minutes at roomtemperature and then refluxed under N₂ in an oil bath for 24 hours. Theresulting mixture was cooled to room temperature and suction-filtered toremove the solid byproduct. The filtrate was extracted with EtOAc (3×100mL). The combined organic layer was washed with H₂O (2×100 mL) and thenwith saturated NaCl (2×150 mL), and subsequently dried with anhydrousNa₂SO₄. Evaporation of the solvent gave crude1-phenyl-2(2-pyridyl)imidazole. The crude product is generally pureenough to use in making redox mediators, although the crude product maybe further purified using a silica gel column and eluting withMeOH/CHCl₃.

[0068] The 1-phenyl-2-(2-pyridyl)imidazole product described above canbe used in the synthesis of a transition metal complex, such as anOsmium complex, in much the same manner 1-methyl-2-(2-pyridyl)imidazolewas used in Example 1 above.

Examples of Further Transition Metal Complexes

[0069] Further transition metal complexes that serve as redox mediatorsaccording to the present invention are provided in Table 2 below, asMediator Nos. 1-13. The redox potentials (E_(1/2) (mV) relative to astandard Ag/AgCl reference electrode in a pH 7 PBS buffer) associatedwith these redox mediators are also provided, where available.

[0070] Also provided in Tables 3 and 4 are various of these redoxmediators and their associated redox potentials and associated slopes,k, of substantially linear plots of collected charge (μC) versus glucoseconcentration (mg/dL) for a given volume (˜315 ηL) of biofluid, such asblood, as further described below. Comparative information for knownredox mediators, namely, Comparative Mediator Nos. I, X, XII and XIII isalso provided. The slope data in Table 3 and Table 4 concerns redoxmediators tested under Condition A and Condition B, respectively, whichreflect different ink lots, as now described.

[0071] That is, these slope data were obtained from individual tests inwhich each mediator and an enzyme mixture were coated on a workingelectrode. The working electrode was made of a conductive ink layeredover a plastic substrate. The working electrode was laminated togetherwith a counter/reference electrode, using standard processing known inthe art. The counter/reference electrode was made of a Ag/AgCl inklayered over a plastic substrate. Variations are routinely observed intest strip sensors made from different ink lots. Thus, in Table 3,Condition A refers to tests conducted using a series of test strips madefrom a single lot, and in Table 4, Condition B similarly refers to testsconducted using a series of test strips made from a single lot,different from that associated with Condition A. Thus, comparisons ofslope data shown in Table 3 and Table 4 should not be made, whilecomparisons of slope data shown within either Table 3 or Table 4 areinstructive as to mediator performance. TABLE 2 Examples of LowPotential Mediators of the Present Invention Redox Potential, E_(1/2)(mV) Mediator No. Structure of Mediator versus Ag/AgCl 1

−164 2

−168 3

−150 4

−172 5

6

7

8

−139 9

−124 10

−117 11

−130 12

−166 13

−88

[0072] TABLE 3 Examples of Low Potential Osmium Mediators and KnownComparative Mediators and Properties Thereof Under Condition A MediatorNo. or Redox Potential, Comparative Structure of Mediator or E_(1/2)(mV) Linear Slope, k Mediator No. Comparative Mediator versus Ag/AgCl(μC/(mg/dL))  1

−164 1.52  2

−168 1.49  3

−150 1.46  4

−172 1.49 11

−130 1.55 I*

−110 1.14 X*

−125 1.05

[0073] TABLE 4 Examples of Low Potential Osmium Mediators and KnownComparative Mediators and Properties Thereof Under Condition B MediatorNo. or Redox Potential, Comparative Structure of Mediator or E_(1/2)(mV) Linear Slope, k Mediator No. Comparative Mediator Versus Ag/AgCl)(μC/(mg/dL)) 8

−139 1.73 9

−124 1.70 X*

−125 1.48 XII*

−74 1.46 XIII*

−97 1.52

[0074] The transition metal complexes of the present invention are wellsuited for electrochemical sensing applications, given their particularelectrochemical properties. For example, as shown above, the redoxpotentials of the mediators are generally low, such as in a range offrom about 0 mV to about −200 mV relative to a Ag/AgCl referenceelectrode. These redox potentials are particularly desirable forelectrochemical sensing applications, being in a range at which thekinetics of the mediators is fast and the electrochemical activity ofpotentially interfering species is minimized. Mediator Nos. 1-13 thusexemplify electrochemically desirable mediators according to the presentinvention.

[0075] The identity of the potentially interfering species justdescribed depends on the particular electrochemical sensing application.Merely by way of example, when the electrochemical sensing applicationconcerns the biofluid, blood, potentially interfering species includeascorbic acid, acetaminophen, and uric acid. Mediator Nos. 1-13exemplify electrochemically desirable mediators that operate atpotentials suitable for minimizing the electrochemical activity of suchpotentially interfering species, while not sacrificing mediatorefficiency.

[0076] Additionally, the transition metal complexes of the presentinvention are particularly effective redox mediators in electrochemicalsensing applications, given their enhanced ability to collect charge atthe working electrode, which in turn enhances the sensitivity of thesensor to the concentration of the analyte being sensed. By way ofexample, in the general operation of an electrochemical biosensor, suchas a glucose sensor, the reduced enzyme, glucose oxidase or glucosedehydrogenase, transfers its electrons to the working electrode via aparticular process. In that process, the oxidized form of the redoxmediator interacts with the reduced enzyme, thereby receiving anelectron and becoming reduced. The reduced mediator travels to thesurface of the working electrode, typically by random diffusion,whereupon it transfers the collected electron to the electrode, therebybecoming oxidized.

[0077] Ideally, because each glucose molecule loses two electrons in theabove-described process, the total amount of electrons or chargecollected at the working electrode should be equal to two times thenumber of glucose molecules oxidized. In practice, however, the totalamount of charge collected is almost always less than the ideal ortheoretical amount because the electrons may be “lost” during transferfrom the enzyme to the electrode. For example, the reduced enzyme maytransfer the electrons to oxygen or other chemical species, rather thanto the redox mediator. An efficient redox mediator should thus competefavorably for electrons from the enzyme.

[0078] Further, ideally, once the redox mediator receives an electronfrom the enzyme, it should not transfer the electron to anotheroxidative species, such as oxygen or other chemicals present in thesensor, before being oxidized on the working electrode. A good mediatorshould thus compete favorably for electrons from the reduced enzyme, asdescribed above, and be substantially chemically inert during its randomdiffusion to the working electrode whereupon it is oxidized.

[0079] An efficient mediator is particularly important incoulometry-based electrochemical biosensing, in which detection of thebioanalyte is based on the total amount of charge collected at theworking electrode for a given volume of biofluid. When greater charge iscollected at the working electrode, the sensor is advantageously moresensitive. For a coulometry-based glucose sensor, for example, thesensitivity of the sensor may be characterized by the slope value of alinear plot of charge versus glucose concentration as defined by theequation y=kx+b, where y is the collected charge in μC for a givenvolume of biofluid, k is the slope in μC/(mg/dL), x is the glucoseconcentration in mg/dL, and b is the intercept based on backgroundcharge. As demonstrated above, mediators of the present invention thathave a negatively charged ligand, such as Mediator Nos. 1-13 that have achloride ligand, have associated slope values that are significantlyhigher (for example, about 28% to about 48% higher per Table 3, andabout 11% to about 18% higher per Table 4) than those of mediators thathave heterocyclic nitrogen-containing ligands surrounding the metalredox center, as exemplified by Comparative Mediator Nos. I, X, XII andXIII.

[0080] The above-described data demonstrate favorable properties oftransition metal complexes that make these complexes particularlydesirable redox mediators. In electrochemical sensing applications, suchas the electrochemical sensing of glucose, the transition metalcomplexes effectively collect electrons from the reduced enzyme andeffectively retain the collected electrons prior to delivering them tothe working electrode.

[0081] As described herein, the transition metal complexes of thepresent invention are usefully employed as redox mediators inelectrochemical sensors. These mediators have very fast kinetics, suchthat electron exchange between such a mediator and the enzyme and/or theworking electrode in the sensor device is rapid, and more particularly,rapid enough to facilitate the transfer of electrons to the workingelectrode that might otherwise be transferred to another electronscavenger, such as oxygen. The electron-transfer efficiency of amediator of Formula 1 is enhanced when L₂ is a negatively chargedligand, such as a chloride ligand, as demonstrated by the desirableslope values, k, listed above for Mediator Nos. 1-13. By way ofcomparison, a mediator having a neutral ligand, L₂, such as aheterocyclic nitrogen-containing ligand, is less able to transferelectrons from the enzyme to the working electrode, as reflected by thelower slope values listed above for Comparative Mediator Nos. I, X, XIIand XIII.

[0082] The transition metal complex mediators of the present inventionare also quite stable in terms of chemical reactivity with respect tochemical species other than the enzyme and the electrode surface. By wayof example, the chemical stability of a mediator of the presentinvention is such that preferably the predominant, or most preferablythe only, reactions in which it participates involves theabove-described, electron-transfer reaction between the mediator and theenzyme and the electrochemical redox reaction at the working electrode.This chemical stability may be enhanced when a mediator of Formula 1,wherein L₂ is a negatively charged monodentate ligand, has a “bulky”chemical ligand, L₁, that spatially or stereochemically shields theredox center, such as Os^(2+/3+), and thereby, reduces undesirablechemical reactivity beyond the fundamentally desired chemical andelectrochemical activity. Mediator Nos. 1-13, above, are particularexamples of such “bulked”, chemically stable mediators of the presentinvention.

[0083] Further by way of example, the thermal and photochemicalstability of a mediator of the present invention is preferably such thatthe mediator is temperature- and light-stable, respectively, undertypical use, storage and transportation conditions. For example,mediators of the present invention may be easily handled under normallighting conditions and may have a shelf life of at least about 18months at about room temperature, and at least about 2 weeks at about57° C. Mediator Nos. 1-13, above, are particular examples of suchthermally and photochemically stable mediators of the present invention.

[0084] Mediators of the present invention have desirable redoxpotentials in a range at which the electron-transfer kinetics isoptimized, or maximized, and the effect of common interfering speciespresent in biofluid is minimized. Mediator Nos. 1-13, above, areparticular examples of mediators of suitable redox potential.

[0085] The transition metal complex mediators of the present inventionalso have desirable solubility properties, generally having a solubilityof greater than about 0.1 moles/liter at 25° C. for a desired solvent,which is typically an aqueous or a water-miscible solvent.Advantageously, one need only adjust the counter ion or ions, X, ofFormula 1, to obtain a desirable solubility for the solvent of choice,be it aqueous or organic.

[0086] In summary, the present invention provides novel transition metalcomplexes that are particularly useful as redox mediators inelectrochemical sensing applications. The preferred redox mediatorsexchange electrons rapidly with enzymes and working electrodes, arestable, are readily synthesized, and have redox potentials that aretailored for the electrooxidation of a variety of analytes, such asthose in various biological fluids within the human body. Whilemediators of the present invention have been described for the most partin terms of glucose sensing, they are useful for the sensing of otheranalytes, such as lactic acid for example. Generally, if the redoxpotential of the enzyme used in a particular analyte-sensing applicationis negative relative to the redox potential of the mediator, themediator is suitable for that analyte-sensing application. Theadvantageous properties and characteristics of the transition metalcomplexes of the present invention make them ideal candidates for use inthe electrochemical sensing of glucose, an application of particularimportance in the diagnosis and monitoring of diabetes in humanpopulations.

[0087] Various aspects and features of the present invention have beenexplained or described in relation to beliefs or theories, although itwill be understood that the invention is not bound to any belief ortheory. Further, various modifications, equivalent processes, as well asnumerous structures to which the present invention may be applicablewill be readily apparent to those of skill in the art to which thepresent invention is directed upon review of the instant specification.Although the various aspects and features of the present invention havebeen described with respect to various embodiments and specific examplesherein, it will be understood that the invention is entitled toprotection within the full scope of the appended claims.

1. A transition metal complex having the formula:

wherein c is a negative, neutral, or positive charge represented by −1to −5, 0, or +1 to +5, respectively; d is an absence or a number ofcounter ions X represented by 0 or 1 to 5, respectively; M is cobalt,iron, osmium, ruthenium, or vanadium; L₁ is a substituted or anunsubstituted heterocyclic nitrogen-containing ligand; L₂ is anegatively charged ligand; and L and L′ are independently selected froma group consisting of:

wherein R′₁ is a substituted or an unsubstituted alkyl, alkenyl, oraryl; R_(a) and R_(b) are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted or unsubstitutedalkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl,or alkyl; R′₃, R′₄ are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted or unsubstitutedalkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl,or alkyl, or a combination of R′₃ and R′₄ forms a saturated orunsaturated 5- or 6-membered ring; and R_(c), and R_(d) areindependently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂,—SH, —OH, —NH₂, or substituted or unsubstituted alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl, or acombination of Rc and Rd forms a saturated or unsaturated 5- or6-membered ring.
 2. The complex of claim 1, wherein R′₁ is a substitutedor unsubstituted C1-C12 alkyl or alkenyl.
 3. The complex of claim 1,wherein R′₁ is methyl.
 4. The complex of claim 1, wherein R′₁ is asubstituted or unsubstituted aryl.
 5. The complex of claim 1, whereinR′₁ is a substituted or unsubstituted phenyl.
 6. The complex of claim 1,wherein R′₁ is a phenyl substituted with a substituent selected from agroup consisting of —Cl, —F, —CN, amino, carboxy, C1-C6 alkyl, C1-C6alkylthio, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6alkylaminocarbonyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, and C1-C6alkylcarboxamido.
 7. The complex of claim 1, wherein R′₃, R′₄, R_(a),R_(b), R_(c), and R_(d) are independently —H or substituted orunsubstituted alkyl.
 8. The complex of claim 1, wherein c is 0, +1, +2,or +3.
 9. The complex of claim 1, wherein X is an anion selected from agroup consisting of halides, sulfates, phosphates, hexafluorophosphatesand tetrafluoroborates.
 10. The complex of claim 1, wherein X ischloride.
 11. The complex of claim 1, wherein d is
 2. 12. The complex ofclaim 1, wherein L₁ is a derivative of any of imidazole, triazole,oxazole, thiazole, and pyrazole.
 13. The complex of claim 1, wherein L₁has the formula:

wherein R₇ is a substituted or an unsubstituted alkyl, alkenyl, or aryl;R₈ is —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂,—SH, —OH,—NH₂, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, oralkyl; and R₉ and R₁₀ are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl, or acombination of R₉ and R₁₀ forms a fused, saturated or unsaturated, 5- or6-membered ring.
 14. The complex of claim 13, wherein R₇ is asubstituted or unsubstituted C1-C12 alkyl or alkenyl.
 15. The complex ofclaim 13, wherein R₉ and R₁₀ form a fused, unsaturated 6-membered ring.16. The complex of claim 13, wherein R₈, R₉ and R₁₀ are independently —Hor substituted or unsubstituted alkyl.
 17. The complex of claim 1,wherein L₁ has the formula:

wherein R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are independently —H, —F, —Cl, —Br,—I, —NO₂, —CN, —CO₂H, —OH, —NH₂, alkoxycarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino,arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino,alkylthio, alkenyl, aryl, or alkyl.
 18. The complex of claim 17, whereinat least one of R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ is an alkyl substituted with—F, —Cl, —Br, —I, alkylamino, dialkylamino, trialkylammonium, alkoxy,alkylthio, aryl, or a reactive group, or an aryl substituted with —F,—Cl, —Br, —I, alkylamino, dialkylamino, alkoxy, alkylthio, aryl, or areactive group.
 19. The complex of claim 17, wherein R₁₁, R₁₂, R₁₃, R₁₄and R₁₅ are independently —H, methyl, C1-C2 alkoxy, C1-C2 alkylamino,C2-C4 dialkylamino, or a C1-C6 alkyl substituted with a reactive group.20. The complex of claim 1, wherein L₂ is selected from a groupconsisting of —CN, —SCN, —OH, halide, alkoxy, alkylthio, and phenoxide.21. The complex of claim 1, wherein L₂ is chloride.
 22. The complex ofclaim 1 having a redox potential of from about 0 mV to about −200 mVrelative to a Ag/AgCl reference electrode.
 23. The complex of claim 1,wherein M is osmium.
 24. The complex of claim 1, wherein at least one ofL, L′, L₁ and L₂ is coupled to a polymeric backbone.
 25. The complex ofclaim 24, wherein the polymeric backbone comprises at least onefunctional group that is a ligand of the complex.
 26. The complex ofclaim 25, wherein the functional group is selected from a groupconsisting of pyridine and imidazole groups.
 27. The complex of claim 1,wherein said complex is a polymeric product of a reaction of a precursorpolymer and a precursor transition metal complex.
 28. A transition metalcomplex having the formula:

wherein c is a neutral or positive charge represented by 0, or +1 to +3,respectively; d is absence or a number of counter anions X representedby 0 or 1 to 5, respectively; M is osmium; L₁ is a substituted or anunsubstituted heterocyclic nitrogen-containing ligand; L₂ is from agroup consisting of —CN, —SCN, —OH, halide, alkoxy, alkylthio, andphenoxide; and L and L′ are independently selected from a groupconsisting of:

wherein R′₁ is a substituted or an unsubstituted C1-C12 alkyl; and R′₃,R′₄, R_(a), R_(b), R_(c), and R_(d) are independently —H or substitutedor unsubstituted alkyl.
 29. The complex of claim 28, wherein X is ananion selected from a group consisting of halides, sulfates, phosphates,hexafluorophosphates and tetrafluoroborates.
 30. The complex of claim28, wherein L₂ is selected from a group consisting of —CN, —SCN, —OH,halide, alkoxy, alkylthio, and phenoxide.
 31. The complex of claim 28,wherein L₁ is a derivative of any of imidazole, triazole, oxazole,thiazole, and pyrazole.
 32. The complex of claim 28, wherein L₁ has theformula:

wherein R₇ is a substituted or an unsubstituted C1-C12 alkyl or alkenyl;and R₈, R₉ and R₁₀ are independently —H or substituted or unsubstitutedalkyl.
 33. The complex of claim 28, wherein L₁ has the formula:

wherein R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ are independently —H, methyl, C1-C2alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 alkylsubstituted with a reactive group.
 34. The complex of claim 28 having aredox potential of from about 0 mV to about −200 mV relative to aAg/AgCl reference electrode.
 35. The complex of claim 28, wherein atleast one of L, L′, L₁ and L₂ is coupled to a polymeric backbone. 36.The complex of claim 35, wherein the polymeric backbone comprises atleast one functional group that is a ligand of the complex.
 37. Thecomplex of claim 36, wherein the functional group is selected from agroup consisting of pyridine and imidazole groups.
 38. The complex ofclaim 28, wherein said complex is a polymeric product of a reaction of aprecursor polymer and a precursor transition metal complex.
 39. A sensorcomprising: a working electrode; a counter electrode; a redox mediatordisposed proximate to the working electrode, the redox mediator havingthe formula:

wherein c is a negative, neutral, or positive charge represented by −1to −5, 0, or +1 to +5, respectively; d is an absence or a number ofcounter ions X represented by 0 or 1 to 5, respectively; M is cobalt,iron, osrnium, ruthenium, or vanadium; L₁ is a substituted or anunsubstituted heterocyclic nitrogen-containing ligand; L₂ is anegatively charged ligand; and L and L′ are independently selected froma group consisting of:

wherein R′₁ is a substituted or an unsubstituted alkyl, alkenyl, oraryl; R_(a) and R_(b) are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted or unsubstitutedalkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl,or alkyl, R′₃, R′₄ are independently —H, —F, —Cl, —Br, —I, —NO₂, —CN,—CO₂H, —SO₃H, —NHNH₂, —SH, —OH, —NH₂, or substituted or unsubstitutedalkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy,alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino,alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl,or alkyl, or a combination of R′₃ and R′₄ forms a saturated orunsaturated 5- or 6-membered ring; and R_(c), and R_(d) areindependently —H, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂,—SH, —OH, —NH₂, or substituted or unsubstituted alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino,dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino,hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl, or acombination of R_(c) and R_(d) forms a saturated or unsaturated 5- or6-membered ring.
 40. The sensor of claim 39, wherein c is a neutral orpositive charge represented by 0, or +1 to +3, respectively; M isosmium; L₂ is selected from a group consisting of —CN, —SCN, —OH,halide, alkoxy, alkylthio, and phenoxide; R′₁ is a substituted or anunsubstituted C1-C12 alkyl; and R′₃, R′₄, R_(a), R_(b), R_(c), and R_(d)are independently —H or substituted or unsubstituted alkyl groups. 41.The sensor of claim 39, further comprising an enzyme disposed proximateto the working electrode.
 42. The sensor of claim 39, wherein the redoxmediator has a redox potential of from about 0 mV to about −200 mVrelative to a Ag/AgCl reference electrode.
 43. The sensor of claim 39,wherein the redox mediator is coupled to a polymeric backbone via atleast one of L, L′, L₁ and L₂.
 44. The sensor of claim 43, wherein thepolymeric backbone comprises at least one functional group that is aligand of the complex.
 45. The sensor of claim 44, wherein thefuinctional group is selected from a group consisting of pyridine andimidazole groups.
 46. The sensor of claim 39, wherein said complex is apolymeric product of a reaction of a precursor polymer and a precursortransition metal complex.
 47. The sensor of claim 39, wherein the redoxmediator is crosslinked on the working electrode.
 48. The sensor ofclaim 39, wherein the redox mediator and an enzyme are crosslinked onthe working electrode.